Tertiary amine compounds for use in immunoassays

ABSTRACT

A reagent for use in immunoassays reduces interference in particle agglutination assays. The reagent contains particles having covalently bound antibodies and a tertiary amine compound of formula (I): 
     N(R 1 —X)(R 2 —Y)(R 3 —Z)  (I). 
     The moieties R 1 , R 2 , and R 3  are independently alkyl or alkyl ether. The moieties X, Y, and Z are independently —OH, —O—R 4 , —S—R 4 , —C(═O)—OH, —C(═O)—OR 4 , or —C(═O)—NHR 4  (R 4  is alkyl).

BACKGROUND OF THE INVENTION

[0001] In testing for analytes such as drug molecules, immunoassays haveproven to be especially useful. In an immunoassay, the interaction of ananalyte, sometimes referred to as an antigen, with a specific receptor,typically an antibody, results in the formation of an antigen-antibodycomplex. This complex can be detected by various measurements, such asradioactivity, fluorescence, light absorption and light scattering. Theresults are then correlated with the presence, absence, and ideally theconcentration of the analyte.

[0002] One type of immunoassay is the particle-based agglutinationimmunoassay, which is based on the binding of an antigen with anantibody, one of which is bound to a particle. The particles employedare often polymer particles, such as polystyrene and poly(methylmethacrylate), which are typically produced by an emulsionpolymerization process. Other particle systems may also be used,including gold particles such as gold nanoparticles and gold colloids;and ceramic particles, such as silica, glass, and metal oxide particles.The binding agent, which is an antigen or an antibody, may be physicallyadsorbed onto the particle; however, greater stability and longershelf-life are obtained when the binding agent is covalently attached.See for example J. L. Ortega-Vinuesa et al. J. Biomater. Sci. PolymerEdn., 12(4), 379-408 (2001).

[0003] Particles having covalently linked binding agents are typicallyprepared by activation of the particles, followed by coupling of thebinding agent to the activated particles. In some instances, theactivation is followed by a coupling of a linking group to the activatedparticles, and the linking group can then be used to tether the bindingagent to the particle. Linking groups include, for example, avidin orstreptavidin and chemical moieties presenting functional groups such asmaleimides and thiols.

[0004] For particles having carboxylate groups bound to the surface,activation is often achieved by contacting the particles with a solutionof a carbodiimide coupling reagent and a succinimide reagent such asN-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sNHS). Thecarboxylate groups on the surface are thus converted into NHS-ester orsNHS-ester groups. Carbodiimide couplers include, for example,N-ethyl-N′-(3-dimethyl-aminopropyl)carbodiimide (EDC);dicyclohexylcarbodiimide (DCC); and diisopropylcarbodiimide (DIC). Abinding agent or a linking group can then be coupled to the particles bymixing the activated particles and the binding agent or linking group inan aqueous mixture. Sensitized particles are formed once a binding agenthas been linked to the surface, whether by contact with activatedparticles or by reaction with particles containing linking groups. Anillustration of a simple version of this process, using NHS as thesuccinimide reagent and sensitizing with an antibody, is given in thefollowing reaction scheme. The sensitized particles produced by thisprocess are then typically treated with a blocking agent, for examplebovine serum albumin (BSA), which serves to react with and quenchremaining NHS or sNHS ester groups.

[0005] When such sensitized particles are mixed in an aqueousenvironment with a sample to be analyzed, the analyte in the sample willspecifically bind to the antibody, which may be present on the particleor as a separate entity in the liquid mixture. This interaction maycause the particles to agglutinate (direct recognition), or it mayhinder an agglutination process (competitive inhibition), depending onthe particular format of the assay. Agglutination is the formation ofclusters of particles having a larger collective size than that of anindividual particle, and can be detected by measuring the change in theabsorbance or the scattering of light by the sample. Ideally, the degreeof agglutination in a particle-based agglutination immunoassay can becorrelated with the amount of antigen in the sample. However,non-specific interactions between the particles and the sample canresult in agglutination or inhibition of agglutination of the particleswhich is unrelated to the antigen-antibody interaction. These unwantedinteractions can cause false positive or false negative results, and canalso lead to inaccurate correlations of the agglutination response tothe concentration of the antigen of interest. All of these undesirableeffects compromise the quality of the assay result, and are collectivelyknown as “interference.”

[0006] Various substances have been reported to reduce interference inagglutination immunoassays by suppressing non-specific interactions. Forexample, U.S. Pat. No. 4,362,531 describes the use of salts such asguanidinium salts, thiocyanate salts, and alkali metal halide salts. Inaddition, U.S. Pat. Nos. 5,506,151 and 5,486,479 describe the use ofprimary, secondary, and tertiary amines such as1-ethyl-3-(3-dimethylaminopropyl)urea (EDU), 3-dimethylaminopropylamine,3-diethylaminopropylamine, dimethylaminopropylchloride, and1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate(CMC). Other substances reported include halogen substituted carboxylicacids (U.S. Pat. No. 4,536,478) and substituted amides (U.S. Pat. No.4,292,038). In general, these additives do not provide an optimumbalance between reducing the interference due to non-specificinteractions and minimizing the reactivity of the additive towards theantibody or antigen of interest. Non-specific interactions between theadditive and the antibody or antigen can also contribute to interferencein the immunoassay.

[0007] It is thus desirable, in particle-based agglutinationimmunoassays, to prevent non-specific interactions between the particlesand components of the sample being analyzed. It is also desirable toprovide additives which can suppress non-specific interactions withinthe immunoassay while avoiding interactions with the antigen orantibody.

SUMMARY OF THE INVENTION

[0008] In one aspect of the invention there is a reagent for use inimmunoassays, comprising a plurality of particles and a tertiary aminecompound of formula (I)

N(R¹—X)(R²—Y)(R³—Z)  (I);

[0009] wherein R¹, R², and R³ are independently selected from the groupconsisting of alkyl and alkyl ether; and X, Y, and Z are independentlyselected from the group consisting of —OH, —O—R⁴, —S—R⁴, —C(═O)—OH,—C(═O)—OR⁴, or —C(═O)—NHR⁴, wherein R⁴ is alkyl. Each of said particlescomprises a surface having been activated by a carbodiimide, and abinding agent linked to the surface through a covalent bond.

[0010] In another aspect of the invention, there is a reagent for use inimmunoassays, comprising a plurality of particles and a tertiary aminecompound of formula (II)

N(R¹—OH)(R²—OH)(R³—OH)  (II);

[0011] wherein R¹, R², and R³ are independently alkyl groups comprisingfrom 1 to 5 carbon atoms; wherein the reagent forms an assay mixturewhen mixed with a sample, such that the tertiary amine compound ispresent in the assay mixture in a concentration of 50 mM or less. Eachof said particles comprises a surface having been activated by acarbodiimide, and a binding agent linked to the surface through acovalent bond.

[0012] In yet another aspect of the invention there is an assay methodfor determining an analyte, comprising combining a sample suspected ofcontaining said analyte with any of the above reagents, and determiningthe presence or amount of said detectable complex as a measure of saidanalyte in said sample. The reagent comprises the antibody of saidanalyte, and the reagent is capable of forming a detectable complex withsaid analyte.

[0013] In yet another aspect of the invention there is provided a testkit, comprising any of the above reagents.

[0014] In yet another aspect of the invention there is provided, in animmunoassay method wherein a sample suspected of containing an analyteis combined with a plurality of particles, each of said particles havinga surface having been activated by a carbodiimide, and a binding agentbound to the surface through a covalent bond; the improvement comprisingadding to the sample, to form an assay mixture, a tertiary aminecompound of formula (I)

N(R¹—X)(R²—Y)(R³—Z)  (I);

[0015] wherein R¹, R², and R³ are independently selected from the groupconsisting of alkyl and alkyl ether; and X, Y, and Z are independentlyselected from the group consisting of —OH, —O—R⁴, —S—R⁴, —C(═O)—OH,—C(═O)—OR⁴, or —C(═O)—NHR⁴, wherein R⁴ is alkyl.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a graph of the dependence of the absorbance at 468 nmdue to bound acid orange 7 dye as a function of particle concentration.

[0017]FIG. 2 is a graph of the dependence of the absorbance at 468 nmdue to bound acid orange 7 dye as a function of particle concentration,with background signal subtracted.

[0018]FIG. 3 is a graph correlating the measurements of gentamicinimmunoassays performed by fluorescence polarization and by particleagglutination without triethanolamine (TEO) present.

[0019]FIG. 4 is a graph correlating the measurements of gentamicinimmunoassays performed by fluorescence polarization and by particleagglutination with TEO present.

DETAILED DESCRIPTION

[0020] The present invention is intended to reduce interference inparticle agglutination immunoassays. Particles which have been activatedwith a carbodiimide and either NHS or sNHS and then sensitized with abinding agent are used in immunoassays in the presence of certaintertiary amine compounds. The tertiary amine compounds used in thepresent invention are remarkably effective in the prevention ofnon-specific interactions between the particles and sample components.The reduction or elimination of non-specific interactions improves theaccuracy of the immunoassay.

[0021] Particle agglutination immunoassays of an analyte utilizeparticles which contain a binding agent on their surface. The antibodyof the analyte undergoes a specific interaction with an analyte in thesample to be analyzed and optionally with a conjugate of the analytealso present in the assay mixture. These interactions affect the degreeof aggregation of the particles, and this aggregation can be monitoredand correlated with the amount of the analyte in the sample. Theagglutination immunoassays may be direct assays, in which the binding ofthe antibody and analyte directly affect the aggregation; or they may becompetitive inhibition assays, in which the analyte competes with aconjugate derivative of the analyte for binding with the availableantibody.

[0022] Analyte refers to the substance, or group of substances, whosepresence or amount thereof in a liquid medium is to be determinedincluding, but not limited to, any drug or drug derivative, hormone,protein antigen, oligonucleotide, hapten, or hapten-carrier complex. Ananalyte analog is any substance, or group of substances, which behavesin a similar manner to the analyte, or in a manner conducive toachieving a desired assay result with respect to binding affinity and/orspecificity of the antibody for the analyte including, but not limitedto, derivatives, metabolites, and isomers thereof.

[0023] Antibody means a specific binding partner of the analyte and ismeant to include any substance, or group of substances, which has aspecific binding affinity for the analyte to the exclusion of othersubstances. The term includes polyclonal antibodies, monoclonalantibodies and antibody fragments.

[0024] A peptide is any compound formed by the linkage of two or moreamino acids by amide (peptide) bonds, usually a polymer of α-amino acidsin which the α-amino group of each amino acid residue (except theNH₂-terminal) is linked to the α-carboxyl group of the next residue in alinear chain. The terms peptide, polypeptide and poly(amino acid) areused synonymously herein to refer to this class of compounds withoutrestriction as to size. The largest members of this class are referredto as proteins.

[0025] A covalent bond is a chemical bond between two species, and mayinvolve single bonds or multiple bonds. The term “covalent” does notinclude hydrophobic/hydrophilic interactions, Hydrogen-bonding, van derWaals interactions, and ionic interactions.

[0026] Any sample that is suspected of containing the analyte can beanalyzed by the method of the present invention. The sample is typicallyan aqueous solution such as a body fluid from a host, for example,urine, whole blood, plasma, serum, saliva, semen, stool, sputum,cerebral spinal fluid, tears, mucus or the like, but preferably isurine, plasma or serum. The sample can be pretreated if desired and canbe prepared in any convenient medium. An aqueous medium is preferred.

[0027] Calibration material means any standard or reference materialcontaining a known amount of the analyte to be measured. The samplesuspected of containing the analyte and the calibration material areassayed under similar conditions. Analyte concentration is thencalculated by comparing the results obtained for the unknown specimenwith results obtained for the standard.

[0028] Particles which may be used in agglutination immunoassays includeany type of particle which are activated using carbodiimide chemistry.Such particles include, for example, polymer particles includingpolystyrene and poly(methylmethacrylate); gold particles including goldnanoparticles and gold colloids; and ceramic particles including silica,glass, and metal oxide particles. See for example C. R. Martin et al.Analytical Chemistry—News & Features, May 1, 1998, 322A-327A.

[0029] These particles may be activated using carbodiimide chemistrydirectly, or they may be activated once their surfaces have beenmodified to contain carboxylate groups. Carboxylate groups can beintroduced to surfaces, for example by hydrolysis reactions, bytreatment with a carboxylating reagent, or by formation ofself-assembled monolayers (SAMs) containing carboxylate groups. See forexample R. G. Chapman et al. J. Am. Chem. Soc., 122, 8303-8304 (2000).Activated particles are then mixed with an antibody, optionally followedby exposure to BSA, to produce sensitized particles. These sensitizedparticles may be further treated with a primary amine compound toprevent covalent interactions between sample components and any residualNHS or sNHS esters on the particle surface. Suitable primary aminesinclude, for example, glycine ethyl ester, 2-(aminoethoxy)ethanol (AEO);2,2′-(ethylenedioxy)bisethylamine (EBE); or4,7,10-trioxa-1,3-tridecanediamine (TTD) as described in co-pendingapplication serial no. ______, attorney docket number 9793-102, entitled“Particles For Immunoassays And Methods For Treating The Same” filedDec. 18, 2001, with inventors C. C. Lawrence et al., the disclosure ofwhich is incorporated herein by reference.

[0030] Analysis of the carbodiimide activation chemistry reveals thattertiary amine functional groups linked to the surface of the particlescan be formed by conversion of the intermediate O-acylisoureaintermediate to an N-acylurea moiety. During the conversion of theparticle-bound carboxylate groups into NHS-esters or sNHS-esters, it isbelieved that the presence of excess EDC can lead to the formation ofN-acylurea moieties on the particle surface as illustrated in thefollowing reaction scheme. These N-acylurea moieties are likely stableduring the subsequent processing steps (sensitization and treatment withprimary amine) and under normal immunoassay conditions.

[0031] The conversion of the O-acylisourea group to the N-acylurea groupcompetes both with the desired esterification between the O-acylisoureaintermediate and NHS or sNHS and with the hydrolysis of the intermediateback to the free carboxylate. The hydrolysis reaction of theO-acylisourea to the carboxylate is believed to predominate over boththe esterification reaction and the N-acylurea forming side reaction.The formation of the N-acylurea moiety may be catalyzed by a secondequivalent of the carbodiimide (N. Nakajima and Y. Ikada, BioconjugateChem., 6, 123-130, (1995)). Typical activation protocols call for NHS tobe added to the particle suspension before EDC is added to initiate thereactions. Once the O-acylisourea intermediate is formed, its rate ofconversion to an N-acylurea moiety is minimized under these conditions.The rate of this side-reaction is more problematic when a high degree ofcarboxylate activation is desired, since this requires the use of alarge number of equivalents of EDC.

[0032] The presence of N-acylurea moieties on the particle surface isundesirable. This group and its linkage to the particle are chemicallystable, and thus can remain on the microparticle throughout all of thesubsequent preparatory steps, including both the sensitization withbinding agent and bovine serum albumin, as well as the quenching of anyresidual NHS-esters or sNHS-esters with a primary amine. Moreover, thetertiary amine group at the terminus of the N-acylurea moiety will beprotonated under the pH conditions of the immunoassay (5<pH<9) andtherefore has the potential to engage in non-specific electrostaticinteractions with appropriately charged or polar components present inthe sample being analyzed.

[0033] The effects of the N-acylurea groups on the accuracy of animmunoassay can be observed by analyzing the interaction of immunoassayparticles with a dye which, like many proteins, can interact with thetertiary amine of the N-acylurea group. Referring to FIG. 1, particleswhich have been activated and treated with a primary amine adsorb thedye on their surface, as measured by the optical absorbance of theparticles. The dye in this case is acid orange 7, which is a dye withaffinity for amino groups which have been protonated under acidic pHconditions. The dye binds to particles which have been subjected to acarbodiimide activation step, and this binding is dependent on theconcentration of particles in the dye—particle mixture. In contrast,significant binding of the dye does not occur with particles which havenot been subjected to the activation procedure. The binding of the dyeis a model for the non-specific interactions that can occur betweenparticles and sample components, resulting in interference.

[0034] The interference generated by the presence of N-acylurea groupson the particle surface can be reduced or eliminated by the presence ofa tertiary amine compound of formula (I) in the immunoassay mixture:

N(R¹—X)(R²—Y)(R³—Z)  (I),

[0035] where R¹, R², and R³ are independently alkyl or alkyl ether; andX, Y, and Z are independently —OH, —O—R⁴, —S—R⁴, —C(═O)—OH, —C(═O)—OR⁴,or —C(═O)—NHR⁴, where R⁴ is alkyl. “Alkyl” refers to a substituted orunsubstituted, straight, branched or cyclic hydrocarbon chain. “Alkylether” refers to an alkyl group containing at least one —C—O—C-bond.Preferably, R¹, R², R³, and R⁴ are independently alkyl groups containingfrom 1 to 5 carbon atoms. More preferably, R¹, R², R³, and R⁴independently contain from 1 to 3 carbon atoms. Preferably, X, Y, and Zare independently —OH or —O—R⁴. More preferably, X, Y, and Z are all—OH.

[0036] Without wishing to be bound by any theory of operation, it isbelieved that the tertiary amine compounds reduce interference byinteracting electro-statically and non-specifically with samplecomponents which would otherwise engage in non-specific electrostaticinteractions with the tertiary amine functionality of the N-acylureamoieties present on the particle surface. The tertiary amine compoundseffectively function as decoys, thus minimizing or eliminating theinteractions between N-acylureas and sample components. The inhibitionof non-specific interactions involving the particles improves theaccuracy of the immunoassay.

[0037] The tertiary amine compounds may be combined in an aqueousmixture with the particles, or they may be combined with the samplebefore the sample is mixed with the particles. The “reagent” added tothe sample to perform the immunoassay thus can be a single componentcontaining particles and the tertiary amine compound; or it can be morethan one component, where each component can be added to the samplealone or in combination. It is preferred that, in the final mixturewhich is analyzed, referred to as the assay mixture, the tertiary aminecompound is present in a concentration of 50 mM or less. Morepreferably, the tertiary amine compound is present in a concentration of25 mM or less. Even more preferably, the tertiary amine compound ispresent in a concentration of 12.5 mM or less. Even more preferably, thetertiary amine compound is present in a concentration of 5 mM or less.

[0038] It is preferred that —R¹—X, —R²—Y, and —R³—Z are independentlyelectron-withdrawing groups. An electron-withdrawing group contains anelement or group of elements at or near its terminus which is moreelectronegative than the element to which the electron-withdrawing groupis attached. In these examples, the electronegative portion isidentified as X, Y, and Z. The effect of an electron-withdrawing groupis to increase the partial positive charge on the central tertiarynitrogen by attracting electron density toward the X, Y, and Z groupsand away from the central nitrogen. A tertiary nitrogen with increasedpartial positive charge has an increased ability to interactelectrostatically with substances which are charged or polar. The —R¹—X,—R²—Y, and —R³—Z groups of the tertiary amine compounds of the presentinvention are not the strongest electron-withdrawing moieties known.However, these groups represent a balance of electronegativity andchemical stability. Other electron-withdrawing groups would likely bereactive towards sample components including the antibody or antigen ofinterest.

[0039] Although the tertiary amine compounds of the present inventioncompete with particle-bound N-acylureas for non-specific interactionwith sample components, they do not interfere with the specificinteraction of the particle-bound antibody with the antigen of interest.In fact, the performance of immunoassays utilizing the tertiary aminecompounds is improved by the lack of interference. FIGS. 3 and 4together illustrate the improved performance of particles of the presentinvention in an immunoassay. FIG. 3 is a graph correlating themeasurements of an immunoassay for gentamicin performed by fluorescencepolarization (FP) with the measurements of an immunoassay for gentamicinperformed by particle agglutination with no added tertiary aminecompound. FIG. 4 is a graph correlating the same FP gentamicinmeasurements with gentamicin measurements from a particle agglutinationimmunoassay using a tertiary amine compound additive, in this case 12.5mM triethanolamine (N(CH₂CH₂OH)₃; TEO). The best-fit line correlatingthe data points should ideally be linear with a slope of 1, a y-axisintercept of zero, and a correlation coefficient (R) of 1. By each ofthese three measures the performance of the assay containing TEO issuperior to that performed without TEO. The presence of the tertiaryamine compound provides a slope of the best-fit line of 1.01, whereasits absence provides a slope of the best-fit line of 1.11. The presenceof the tertiary amine compound provides an intercept of 0.05, whereasthe absence provides an intercept of −0.11. The tertiary amine compoundprovides an R value of 0.985, whereas the absence of tertiary aminecompound provides an R value of 0.976. These comparative results alsoare visually apparent by comparison of the ideal line with the best-fitline calculated from the data.

[0040] A comparison of tertiary amines as additives to particleagglutination immunoassays reveals that the tertiary amine compounds ofthe present invention provide for more accurate results than do othertertiary amines. Referring to Example 3 and Table A herein, whichcompare the parameters of best fit lines for a variety of tertiaryamines in a gentamicin immunoassay, the best fit line for TEO has themost favorable combination of values, not only for the slope, but alsofor the intercept and the R value. Although other tertiary amines haveone or two favorable values for the best fit parameters, TEO hasfavorable values for all three. For example, triethylamine has interceptand R values similar to those of TEO; however triethylamine has a slopevalue farther from 1.0 than does TEO.

[0041] These results demonstrate that an improved performance of aparticle based immunoassay can be obtained by including a tertiary aminecompound of the present invention in the assay mixture. The inclusion ofa tertiary amine compound may be used alone or in combination with othertechniques for reducing interference in an immunoassay. In optimizingthe performance of particle agglutination immunoassays, it may bepreferred to include a tertiary amine compound of the present inventionin the assay mixture, and also to use sensitized particles which havebeen treated with a primary amine compound, as described in the abovementioned co-pending application serial no. ______, attorney docketnumber 9793-102, entitled “Particles For Immunoassays And Methods ForTreating The Same” filed Dec. 18, 2001, with inventors C. C. Lawrence etal. In some cases, the use of sensitized particles treated with aprimary amine, such as glycine ethyl ester, AEO, EBE and TTD, may besufficient to reduce the interference of the immunoassay to the desiredlevel. The use of either the primary amine particle treatment or thetertiary amine compound additive, alone or in combination, can bedetermined empirically to determine if one technique is better than theother or if the combination yields the best results.

[0042] Without wishing to be bound by any theory, it is believed thatfailure to compete with the N-acylurea group in this fashion can insteadresult in the interaction of the N-acylurea with protein components ofthe biological fluid during the immunoassay. This in turn interfereswith the kinetics and/or thermodynamics of the particle agglutinationprocess, both in the case of biological fluid samples which contain thetarget analyte and those samples which do not, leading in both instancesto erroneous assay results.

[0043] Various ancillary ingredients will frequently be employed in anassay in accordance with the present invention. For example, bufferswill normally be present in the assay medium, as well as stabilizers forthe assay medium and the assay components. Frequently, in addition tothese additives, additional proteins may be included, such as albumin;or surfactants may be included, particularly non-ionic surfactants andthe like.

[0044] The tertiary amine compound may, along with other components ofthe immunoassay system, be packaged in a kit useful for convenientlyperforming the assay methods for the determination of an analyte. Toenhance the versatility of the subject invention, components can beprovided in packaged combination, in the same or separate containers, inliquid or lyophilized form so that the ratio of the components providesfor substantial optimization of the method and assay. The components mayeach be in separate containers, or various components can be combined inone or more containers depending on the cross-reactivity and stabilityof the components. Preferably, the sensitized particles containing thebinding agent and the tertiary amine are in the same container, suchthat they are added to the sample as a single liquid mixture.

[0045] For example, a test kit may contain, in packaged combination, atertiary amine compound, an antibody specific for a particular analyte,a sensitized particle containing the antibody or a derivative of theanalyte, an analog or derivative of the antibody, or a conjugatedderivative of the analyte. Specific kit combinations include a firstpackage containing the tertiary amine and the antibody together with asecond package containing the particle containing the analytederivative; a first package containing the tertiary amine and theparticle containing the antibody together with a second packagecontaining the conjugated analyte derivative; and a single packagecontaining the tertiary amine, the particle containing the antibody, andthe conjugated analyte derivative. The kit may also comprise one or morecalibrators comprising a known amount of the analyte. Such a test kitmay provide reagents for an assay with enhanced clinical sensitivity forthe analyte and structurally related compounds.

EXAMPLES

[0046] The following examples are provided by way of illustration andshould not be seen as limiting the scope of the present invention.

[0047] Latex particles having an average diameter of 201 nanometers(nm), a surface area of 28.4 square meters per gram (m²/g), andcontaining 0.21 milliequivalents (meq) surface carboxylate groups pergram of latex were obtained from SERADYN (Indianapolis) and used withoutfurther characterization. Microparticle agglutination immunoassays wereperformed on a HITACHI 717 analyzer (ROCHE DIAGNOSTICS CORPORATION,Indianapolis) and their performance assessed with reference to resultsfrom ROCHE fluorescence polarization (FP) immunoassays which wereconducted in parallel on an INTEGRA 700 analyzer (ROCHE DIAGNOSTICSSYSTEMS). ROCHE FP calibrators were used to construct a calibrationcurve for the microparticle-based assay. Resuspension of latex pelletswas effected using a ULTRASONIC HOMEGENIZER-4710 SERIES sonicator(COLE-PARMER, Vernon Hills, Ill.) at 25-50% output power whilemaintaining the sample on ice and latex monodispersity was assessed on aCOBAS MIRA analyzer (ROCHE DIAGNOSTICS SYSTEMS) by light absorption atmultiple wavelengths.

[0048] Solvents and buffers were obtained from FISHER SCIENTIFIC(Suwanee, Ga.). All other reagents were obtained from ALDRICH(Milwaukee, Wis.) or from FLUKA and were used as received.

Example 1 Formation of N-acylurea Groups During Carbodiimide Activation

[0049] To a suspension of 50 ml of 1% (w/v) latex in 10 mM2-morpholinoethanesulfonic acid (MES) having a pH of 5.0, was added 4.78ml a freshly prepared aqueous solution of 0.22 M NHS, followed by 4.03ml of a freshly prepared aqueous solution of 0.26 M1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). After incubationat room temperature for 2 hr, the suspension was centrifuged (15,000×g,30 min), and the latex resuspended in 25 ml of 50 mM3-morpholinopropanesulfonic acid (MOPS) having a pH of 8.0. To thissuspension was added 25 ml of 0.84M 2-(aminoethoxy)ethanol (AEO) in 50mM MOPS at pH 8.0. After incubation at room temperature for 2 hr, thelatex was centrifuged (15,000×g, 30 min), resuspended in 50 ml of 50 mMMOPS at pH 7.0, and centrifuged again. This process was repeated threemore times. The final latex pellets were resuspended in 50 mM MES at pH5.0 and stored as 2% (w/v) suspensions at 4° C.

[0050] Incubations were performed on 1 ml samples containing 0.05-1.0%(w/v) of the latex prepared above and 2 mM acid orange 7 dye, which wasadded as a 50 mM aqueous stock solution. The pH of the sample wasadjusted to 3.0 with 1 M HCl, and the samples were incubated at roomtemperature overnight. Control incubations were performed using latexwhich had not been subjected to the activation procedure with EDC andNHS. The latex was collected by centrifugation (15,000×g, 30 min),resuspended in 1 ml H₂O (pH 3.0), and centrifuged again to removeunbound dye. This procedure was repeated five more times. The resultingpellet was resuspended in 1 ml of 30% (v/v) ethanolamine in water andcentrifuged once more. The absorbance of the supernatant liquid at 468nm was then measured.

[0051] The dependence of the absorption at 468 nm as a function ofparticle concentration is shown in the graphs of FIG. 1. The binding ofacid orange 7 to the non-activated latex particles was taken as thebackground binding, and subtraction of these values from the bindingobserved with the activated-quenched latex at each latex concentrationyields the data shown in FIG. 2. From the slope of the best-fit line(R²=0.998) and the extinction coefficient of the dye, which had beendetermined as ε₄₆₈=13,200 M⁻¹cm⁻¹, on average one in every 38 of thecarboxylate residues on the latex surface was converted to an N-acylureamoiety during the activation—esterification procedure. This calculationassumes a 1:1 stoichiometry of interaction between an N-acylurea moietyand an acid orange 7 molecule. Such an assay can thus be used to monitorand optimize activation conditions of carboxylic acid groups bycarbodiimides.

Example 2 Use of Triethanolamine as an Additive to Improve ImmunoassayPerformance

[0052] Latex agglutination immunoassays were performed using latexparticles which had been sensitized with a gentamicin monoclonalantibody and treated with AEO. Immunoassays were performed in theabsence of triethanolamine (TEO) and in the presence of TEO at finalconcentrations in the range of 2.5-15 mM. The gentamicin content of eachserum sample tested was determined by the commercially available RocheFP gentamicin immunoassay which uses the same gentamicin monoclonalantibody as the latex-based assay. The FP reference immunoassays and thelatex agglutination immunoassays were performed in parallel to avoidsample degradation. The inclusion of TEO in the latex immunoassay bufferdid not significantly affect the quality of the calibration curveobtained.

[0053]FIGS. 3 and 4 show correlation graphs of the microparticle-basedimmunoassays either in the absence of TEO or in the presence of 12.5 mMTEO, with the Roche FP immunoassay. Inclusion of TEO as an additive hasa dramatic effect on the performance of the latex agglutinationimmunoassay. Relative to the control experiment (no TEO), the slope ofthe best-fit line decreases from 1.11 to 1.01, the y-axis interceptchanges from −0.11 to 0.05 and the R correlation coefficient increasesfrom 0.976 to 0.985. Thus the three parameters move closer to theoptimal values of 1.00, 0.00 and 1.000 respectively, as is visuallyapparent from the merging of the best-fit line (solid line) to thetarget line (dotted line (slope=1, intercept=0)). A similar beneficialeffect of including TEO in the microparticle based assay formulation wasseen when the latex was tested with a set of serum samples which hadtested negative for gentamicin by the Roche FP immunoassay. When thefinal assay mixture contained 12.5 mM TEO, the mean apparent gentamicinconcentration in this sample set was −0.32 μg/ml compared to 0.42 μg/mlin the absence of TEO. The desired lower limit of detection of thisassay is 0.17 μg/ml. Optimization studies suggested that a final TEOconcentration of 5 mM was sufficient for the benefits afforded by thisadditive to be fully realized.

Example 3 Comparative Results of Immunoassays Using Tertiary Amines

[0054] Latex agglutination immunoassays for gentamicin were performed asin Example 2, but using a variety of tertiary amines. For eachimmunoassay, 12.5 mM of a tertiary amine was present. The tertiaryamines examined were TEO and the following compounds, previouslydescribed in U.S. Pat. Nos. 5,506,151 and 5,486,479:

[0055] 1-ethyl-3-(3-dimethylaminopropyl)urea (EDU),

[0056] 3-dimethylaminopropylamine,

[0057] 3-diethylaminopropylamine,

[0058] dimethylaminopropylchloride, and triethylamine.

[0059] The parameters of the best fit lines for each set of data aregiven in Table A. The tertiary amine having the slope closest to one wasTEO. The tertiary amines having an intercept closest to zero were EDU,triethylamine and TEO. The tertiary amines having an R value closest toone were dimethylaminopropylchloride and TEO. TABLE A Tertiary AmineSlope Intercept R Control 1.108 −0.111 0.976 EDU 1.094 −0.046 0.9803-dimethylamino- 1.098 −0.211 0.986 propylamine 3-diethylamino- 1.040−0.099 0.983 propylamine dimethylamino- 1.030 −0.178 0.985propylchloride triethylamine 1.031 0.040 0.983 triethanolamine 1.0050.049 0.985 (TEO)

1. A reagent for use in immunoassays, comprising: a plurality ofparticles; each of said particles comprising a surface having beenactivated by a carbodiimide; a binding agent linked to the surfacethrough a covalent bond; and a tertiary amine compound of formula (I)N(R¹—X)(R²—Y)(R³—Z)  (I);wherein R¹, R², and R³ are independentlyselected from the group consisting of alkyl and alkyl ether; and X, Y,and Z are independently selected from the group consisting of —OH,—O—R⁴, —S—R⁴, —C(═O)—OH, —C(═O)—OR⁴, or —C(═O)—NHR⁴, wherein R⁴ isalkyl.
 2. The reagent of claim 1, wherein R¹, R², R³ and R⁴ areindependently alkyl groups comprising from 1 to 5 carbon atoms.
 3. Thereagent of claim 1, wherein X, Y, and Z are independently selected fromthe group consisting of —OH and —O—R⁴.
 4. The reagent of claim 1,wherein R¹, R², and R³ are independently alkyl groups comprising from 1to 5 carbon atoms; and X, Y, and Z are —OH.
 5. The reagent of claim 1,wherein the tertiary amine compound is triethanolamine.
 6. The reagentof claim 1, wherein the reagent forms an assay mixture when mixed with asample; and wherein the tertiary amine compound is present in the assaymixture in a concentration of 50 mM or less.
 7. The reagent of claim 6,wherein the tertiary amine compound is present in the assay mixture in aconcentration of 25 mM or less.
 8. The reagent of claim 6, wherein thetertiary amine compound is present in the assay mixture in aconcentration of 12.5 mM or less.
 9. The reagent of claim 6, wherein thetertiary amine compound is present in the assay mixture in aconcentration of 5 mM or less.
 10. The reagent of claim 1, wherein theparticles further comprise the reaction product of a succinimide esterand a primary amine compound on the surface.
 11. The reagent of claim10, wherein the primary amine compound is selected from the groupconsisting of glycine ethyl ester; 2-(aminoethoxy)ethanol;2,2′-(ethylenedioxy)bisethylamine; and4,7,10-trioxa-1,3-tridecanediamine.
 12. The reagent of claim 1, whereinthe plurality of particles and the tertiary amine compound are presentin a single liquid mixture.
 13. A reagent for use in immunoassays,comprising: a plurality of particles; each of said particles comprisinga surface having been activated by a carbodiimide; a binding agentlinked to the surface through a covalent bond; and a tertiary aminecompound of formula (II) N(R¹—OH)(R²—OH)(R³—OH)  (II);wherein R¹, R²,and R³ are independently alkyl groups comprising from 1 to 5 carbonatoms; wherein the reagent forms an assay mixture when mixed with asample, such that the tertiary amine compound is present in the assaymixture in a concentration of 50 mM or less.
 14. The reagent of claim13, wherein the tertiary amine compound is triethanolamine.
 15. Thereagent of claim 13, wherein the particles further comprise the reactionproduct of a succinimide ester and a primary amine compound on thesurface; wherein the primary amine is selected from the group consistingof glycine ethyl ester; 2-(aminoethoxy)ethanol;2,2′-(ethylenedioxy)bisethylamine; and4,7,10-trioxa-1,3-tridecanediamine.
 16. The reagent of claim 13, whereinthe plurality of particles and the tertiary amine compound are presentin a single liquid mixture.
 17. An assay method for determining ananalyte, comprising: combining a sample suspected of containing saidanalyte with the reagent of claim 1, the reagent comprising the antibodyof said analyte, and the reagent capable of forming a detectable complexwith said analyte; and determining the presence or amount of saiddetectable complex as a measure of said analyte in said sample.
 18. Anassay method for determining an analyte, comprising: combining a samplesuspected of containing said analyte with the reagent of claim 4, thereagent comprising the antibody of said analyte, and the reagent capableof forming a detectable complex with said analyte; and determining thepresence or amount of said detectable complex as a measure of saidanalyte in said sample.
 19. An assay method for determining an analyte,comprising: combining a sample suspected of containing said analyte withthe reagent of claim 6, the reagent comprising the antibody of saidanalyte, and the reagent capable of forming a detectable complex withsaid analyte; and determining the presence or amount of said detectablecomplex as a measure of said analyte in said sample.
 20. An assay methodfor determining an analyte, comprising: combining a sample suspected ofcontaining said analyte with the reagent of claim 13, the reagentcomprising the antibody of said analyte, and the reagent capable offorming a detectable complex with said analyte; and determining thepresence or amount of said detectable complex as a measure of saidanalyte in said sample.
 21. A test kit, comprising the reagent ofclaim
 1. 22. A test kit, comprising the reagent of claim
 4. 23. A testkit, comprising the reagent of claim
 6. 24. A test kit, comprising thereagent of claim
 13. 25. In an immunoassay method wherein a samplesuspected of containing an analyte is combined with a plurality ofparticles, each of said particles having a surface having been activatedby a carbodiimide, and a binding agent bound to the surface through acovalent bond; the improvement comprising: adding to the sample, to forman assay mixture, a tertiary amine compound of formula (I)N(R¹—X)(R²—Y)(R³—Z)  (I);wherein R¹, R², and R³ are independentlyselected from the group consisting of alkyl and alkyl ether; and X, Y,and Z are independently selected from the group consisting of —OH,—O—R⁴, —S—R⁴, —C(═O)—OH, —C(═O)—OR⁴, or —C(═O)—NHR⁴, wherein R⁴ isalkyl.
 26. The method of claim 25, wherein R¹, R², R³ and R⁴ areindependently alkyl groups comprising from 1 to 5 carbon atoms; and X,Y, and Z are —OH.
 27. The method of claim 25, wherein the tertiary aminecompound is present in the assay mixture in a concentration of 50 mM orless.
 28. The method of claim 25, wherein the adding to the samplecomprises: combining the tertiary amine with the particles to form aparticle mixture; and combining the particle mixture with the sample.